The objective of this research is to investigate the role of highly phosphorylated nucleotides and cyclic nucleotides in liver growth. We have so far found that: 1) The highly phosphorylated compounds ppGpp and pppGpp, which are pleiotypic regulators in bacteria, are not functionally significant in either normal or regenerating livers. 2) The levels of another putative pleiotypic regulator, Ap4A, appear to be twice normal in regenerating livers. 3) Two novel highly phosphorylated compounds, each having a modified adenosine as the nucleoside and differing by one phosphate, are present in both normal and regenerating livers. Their relation to growth has not been firmly established, apparently because of their lability in in vivo experiments. 4.) The actively turning over fractions of the endogenous pools of four cyclic nucleotides have been determined by an innovative procedure involving labeling with 32Pi; cAMP values rise in regenerating livers whereas cGMP remains unchanged. No significant incorporation of isotope into cCMP or cUMP occurs, despite highly active labeling of their immediate precursors, the nucleoside triphosphates; the results suggest these two cyclic pyrimidine nucleotides have very low rates of turnover and are therefore of little importance in liver growth. We now propose to extend this research to an in vitro system--primary hepatocyte cultures--from which we have already shown both Ap4A and the above-mentioned novel nucleotides can be readily isolated. The advantages of this system are the controlability of the cellular environment and the ease of isotopic labeling. We will examine the changes in nucleotide concentration and turnover in such cultures, not only in regenerating liver cells but also in cells induced to proliferate by variation of their nutritional or hormonal milieu--either in vivo before cell isolation or subsequently in vitro. The technique of high pressure liquid chromatography (HPLC) which we are beginning to apply, will introduce wholly new dimensions of speed, accuracy, sensitivity, and resolution into this study.